Characterization of a nervous system-specific promoter for growth-associated protein 43 gene in Medaka (Oryzias latipes).
Genes expressed by neurons are controlled by specific, interacting cis-regulatory elements and trans-acting factors within their promoters. In the present study, we asked whether the transcriptional machinery regulating neuron-specific gene expression was conserved in evolution. We identified a GAP-43 homolog in Medaka (Oryzias latipes), and analyzed its expression during various stages of development. Compared with the amino acid sequences of GAP-43 homologs in other vertebrates, the amino-terminus of GAP-43 was highly conserved evolutionarily, but the carboxy-terminus exhibited significant variability. Expression of GAP-43 predominantly occurred in cells of the central and peripheral nervous systems as determined by in situ hybridization and by RT-PCR. Expression of GAP-43 increased throughout development and significant levels continued to be expressed into adulthood. We also showed that a proximal approximately 2.0 kbp fragment in the 5'-flanking region had promoter activity as determined by in vivo reporter assays. Furthermore, based upon computational analysis of transcription factor binding sites and an in vivo reporter analysis using sequentially deleted promoters, we demonstrated that cis-regulatory elements for neuronal expression were widely distributed in this region. In mammals, a TATA-box, E-box and neuronal repressive elements have been thought to contribute to neuronal expression. However, these features were not found in the orthologous region of the Medaka GAP-43 promoter. Our results suggest that the arrangement of cis-regulatory elements of the GAP-43 ortholog in Medaka is different from that in mammals, yet maintains neuron-specific regulation.
Fujimori
Kazuhiro E
KE
Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Nakoji 3-11-46, Amagasaki, Hyogo 661-0974, Japan. k-fujimori@aist.go.jp
Kawasaki
Takashi
T
Deguchi
Tomonori
T
Yuba
Syunsuke
S
D024506:5' Flanking Region / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D029941:Fish Proteins / Q000235:genetics*; D019922:GAP-43 Protein / Q000145:classification / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D017403:In Situ Hybridization; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009420:Nervous System / Q000196:embryology / Q000254:growth & development / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D009990:Oryzias / Q000196:embryology / Q000235:genetics* / Q000254:growth & development; D010802:Phylogeny; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection
D029941:Fish Proteins; D019922:GAP-43 Protein; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins